We have isolated and characterized the complete primary structure of a new member of the tissue inhibitor of metalloproteinase family (TIMP family) which we refer to as TIMP-2. Recent studies have shown that all cells studied to date which secrete the 72 kDa type IV collagenase enzyme secrete this enzyme as a complex with TIMP-2. The majority greater than 95% of secreted 72 kDa gelatinase is found in complexed form as most cells tested secrete 2-4 fold as free TIMP-2. Our studies have shown that TIMP- 2 transcript ion is regulated independently of both TIMP-1 and the 72 kDa type IV collagenase enzyme. We have also demonstrated that TIMP-2 is anti-angiogenic. The mechanism for this effect is two fold; through inhibition of endothelial cell proliferation and blocking endothelial cell-mediated matrix proteolysis. TIMP-2 inhibits tumor cell invasion through reconstituted basement membranes in vitro, and this inhibitor demonstrates erythroid potentiating activity (EPA) . TIMP-2 inhibits proteolytic opening of the blood brain barrier in hemorrhagic stroke models. TIMP-2 genomic clones have been obtained and partial sequencing has identified two introns in the 3' end of the gene. The gene appears to be single copy and is localized on human chromosome 17q25. We have examined the TIMP-2 protein structure and have localized the metalloprotease inhibitory domain to the N-terminal half of the molecule. Further sublocalization has been attempted using a synthetic peptide approach as well as protein crystallization for x-ray diffraction and NMR- spectroscopy.